RU2295562C1 - Bacterium strain bacillus spp. kr-083 as plant protection agent against phytopathogenic microorganism and for growth stimulation thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is new strain of rhizosphere bacteria bacillus spp. KR-083 as plant protection agent against phytopathogenic microorganism and for growth stimulation thereof. Claimed strain is isolated from South Corey rice roots.

EFFECT: simultaneous plant protection agent against phytopathogenic microorganism and plant growth stimulation.

1 ex, 6 tbl

Description

The invention relates to biotechnology and relates to a new strain of rhizospheric bacteria Bacillus spp. KR-083 as a means to protect agricultural plants from phytopathogenic microorganisms and stimulate their growth.

The invention “Biological product Phytosporin for plant protection against diseases” is known and described according to the patent of the Russian Federation No. 2099947 with priority dated 11/15/1996 addressed to the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine (UA) and the Scientific and Production Association "Bashkir" (RU), publication 12/27/1997

The basis of the biological product "Fitosporin" is a bacterial strain Bacillus Subtilis VNIISKHM 128 with a cell concentration of 10 ⁹ -10 ¹⁰ per 1 ml of saline in an amount of 92-98 vol. % and filler in an amount of 2-8 vol. %

Moreover, the strain Bacillus Subtilis VNIISKHM 128 is characterized by high antagonistic activity against phytopathogenic bacteria and fungi, which allows it to be used to protect various types of agricultural, ornamental, and woody plants (prototype).

The objective of the claimed invention is to identify a strain suitable for use in agriculture, not only to protect plants from phytopathogenic microorganisms, but also at the same time to stimulate their growth.

This problem is solved due to the fact that a strain of rhizospheric bacteria Bacillus spp is used to protect agricultural plants from phytopathogenic microorganisms and simultaneously stimulate their growth. KR-083.

The abbreviation “spp” in the name of the strain means that the bacteria belong to the genus Bacillus, but are not identified by type, because have significant differences with existing known species.

The strain of rhizospheric bacteria Bacillus spp. KR-083 was isolated from the roots of rice growing in the Iengnam province of the Republic of South Korea and deposited under registration number “148 VNIISKHM-D” dated July 14, 2005.

Cultivation medium: potato agar (200 g of potato, glucose 10 g, agar 20 g per 1 liter of medium). Temperature 28 ° C, pH 6.8-7.0, microaerophile. The cultivation time at 28 ° C is 48 hours.

The strain is characterized by the following morphological-cultural and physiological-biochemical characteristics.

The cells are in the form of regular rods with rounded ends and a monopolar peritrichous arrangement of flagella. The cell size is (1.5-2.0) × (0.8-1.0) microns. After 24 hours of growth, an accumulation of poly-β-hydroxybutyrate is observed in the liquid nutrient medium. The strain forms spores, positively stained by Gram. Growth in a liquid and semi-liquid nutrient medium is microaerophilic, respiratory metabolism.

On potato-dextrose agar forms round, opaque, colorless, mucous colonies with smooth edges. The diameter of the colonies is 3-5 mm. The optimum temperature for growth is (28-37) ° С. At temperatures above 45 ° C and less than 11 ° C, growth is slow. The optimal pH of the medium is 6.8; growth also occurs at pH from 5.5 to 7.5.

Strain KR-083 is able to fix molecular nitrogen, does not hydrolyze casein and starch, slowly coagulates litmus milk with discoloration of litmus. The strain observed growth at 4 ° C, the presence of 7% NaCl and 0.001% lysozyme. The strain has denitrifying activity to nitrogen gas.

The KR-083 strain uses glucose, arabinose, xylose, lactose, galactose, fructose, sucrose, maltose, mannitol to form acid, as well as rhamnose, dextrin and starch to form alkali as the sole carbon source.

It was revealed and experimentally established that the strain KR-083 exhibits antagonistic properties in relation to pathogens of agricultural diseases - phytopathogenic bacteria and fungi:

- for winter and spring wheat - the strain is effective against powdery mildew (Erysiphe graminis), brown rust (Puccinia recondita), Fusarium root rot (Fusarium culmorum);

- for spring barley - effective against moldy seeds (Penicillium, Alternaria), root rot (Bipolaris sorokiniana);

- for white cabbage and cauliflower - effective against vascular bacteriosis (Xanthomonas campestris), blackleg (Rhizoctonia solani), pitious root rot (Pythium irregulare);

- for potatoes - effective against late blight (Phytophtora infestans), rhizoctonia (Rhizoctonia solani), fusarium wilt (Fusarium oxysporum);

- for grapes - effective against mildew (Plasmopara viticola), oidium (Uncinula necator), gray rot (Botrytis cinerea).

Table 1, 2 presents comparative results on the antagonistic activity of two strains of bacteria: Bacillus subtilis VNIISXM 128 (producer of the biological product Fitosporin) and the claimed strain of Bacillus spp. KR083, in relation to phytopathogenic microorganisms.

Table 1.
Antagonistic activity of strains of rhizospheric bacteria in relation to phytopathogenic bacteria Strain Zone of growth inhibition, mm Erwinia carotovora A-1 Pseudomonas syringiae 8300 Pseudomonas syringiae 2314 Erwinia carotovora 3391 Clavibacter michiganense 17-1 Bacillus subtilis 128 (Fitosporin) 14.2 ± 1.3 18.9 ± 1.6 11.8 ± 0.9 28.8 ± 2.1 16.0 ± 1.2 Bacillus spp. KR-083 (claimed) 27.0 ± 1.8 26.0 ± 2.1 18.0 ± 1.3 32.0 ± 2.8 28.0 ± 2.2

From the data shown in table 1, it is seen that the claimed bacterial strain Bacillus spp. KR-083 has a greater antagonistic activity against phytopathogenic bacteria in comparison with the prototype - strain Bacillus subtilis 128 VNIISCHM - producer of the biological product Fitosporin.

Table 2.
Antagonistic activity of strains of rhizospheric bacteria in relation to phytopathogenic fungi. Strain Zone of growth inhibition, mm Phytophtora capsici Rhizoctonia solani Fusarium culmorum Fusarium solani Pythium spp. Bacillus subtilis 128 (Fitosporin) 26.5 ± 2.2 16.7 ± 1.8 16.2 ± 1.3 16.4 ± 1.3 22.1 ± 2.5 Bacillus spp. KR-083 (claimed) 36.1 ± 2.8 30.7 ± 1.7 34.0 ± 4.4 23.7 ± 1.7 28.1 ± 2.9

From the data shown in table 2, it is seen that the claimed bacterial strain Bacillus spp. KR-083 has a greater antagonistic activity against phytopathogenic fungi compared to the prototype - strain Bacillus subtilis 128 VNIISCHM - producer of the biological product Fitosporin.

For the development of plants, they need nitrogen.

Nitrogen fixation is the incorporation of air nitrogen into a compound available for use by plants and other organisms. Biological nitrogen fixation is carried out by nitrogen-fixing microorganisms. In this regard, the nitrogen-fixing activity of the strain is an important characteristic that affects the stimulation of plant growth.

The claimed bacterial strain Bacillus spp. KR-083 has nitrogen fixing properties, i.e. growth-stimulating properties, in contrast to the prototype - a bacterial strain Bacillus subtilis 128 VNIISCHM, which nitrogen does not fix.

Therefore, for comparison (per standard) of the nitrogen-fixing activity of the bacterial strain Bacillus spp. KR-083 was taken a pure culture of the rhizobacteria Herbas-pirillum seropedicae ATCC 35892, also isolated from the roots of rice (a type strain stored in the American Type Culture Collection).

Table 3 presents data on the nitrogen-fixing activity of the bacterial strain Bacillus spp. KR-083 compared to the standard - nitrogen-fixing bacterium Herbaspirillum seropedicae ATCC 35892 (a type strain stored in the American Type Culture Collection).

Table 3.
Nitrogen-fixing (acetylene reductase) activity of pure cultures of rhizobacteria isolated from rice roots Strain Azotfiksirujushchej (atsetilenreduktaznaya) activity (nmol C ₂ H _4/4 ml medium) after 12 hours After 24 h after 48 hours after 72 hours after 96 hours Herbaspirillum seropedicae ATCC 35892 (reference) 0.93 7.89 170.93 1025.44 1726.75 Bacillus spp. KR-083 (claimed) 0.45 21.48 249.68 635.09 943.61

From the data shown in table 3, it is seen that the claimed bacterial strain Bacillus spp. KR-083 at the initial stage (24-48) hours has a higher nitrogen-fixing activity compared to the standard - a typical strain of nitrogen-fixing bacteria Herbaspirillum seropedicae ATCC 35892, isolated, as well as the claimed strain, from rice roots.

Growth-promoting activity of a strain of Bacillus spp. KR-083 and strain Bacillus subtilis VNIISKHM 128 was determined by two methods:

a) by the method of stimulation of the roots of corn seedlings;

b) the method of biotest with chlorella.

a) Determination of the growth-promoting activity of the strains by the method of stimulation of the roots of corn seedlings.

Corn seeds were washed in running water for 2 hours, placed in plastic trays with moist filter paper with seedlings up, covered with wet filter paper and placed for germination in an incubator at 28 ° C. After 48 hours, seedlings 1-1.5 cm long were selected.

The tips of the roots of corn 2-3 mm were immersed in a bacterial suspension of the corresponding strains of bacteria (for example, in a working culture obtained by the method described below) of various concentrations for 1.5 hours.

As a control, a sterile nutrient medium was used — the cultivation medium of the Bacillus spp strain. KR-083. Then the corn seedlings were placed in plastic trays with moistened filter paper so that the ends of the roots were on the same starting line. The seedlings were covered with wet filter paper and placed in an incubator at 28 ° C. After 24 hours, the roots of the seedlings that crossed the starting line were cut off and their length was measured. Growth-stimulating activity of the compared strains of Bacillus spp. KR-083 and Bacillus subtilis VNIISHM 128 were calculated in relation to the control seedlings.

b) Determination of the growth-promoting activity of the strains by biotest with chlorella.

A method for assessing the growth-stimulating activity of rhizobacteria using the alga Chlorella vulgaris was developed at the VNIISHM. The strain Chlorella vulgaris 157 (obtained from Prof. B.V. Gromov, St. Petersburg State University) was grown on a modified Khata medium (KNO ₃ - 0.1 g / l, KN ₂ PO ₄ - 0.07 g / l, MgSO ₄ × 7H ₂ O - 0.033, peptone - 1 g / l, glucose - 1 g / l, yeast extract - 0.1 g / l, agar - 10 g / l, pH 6.5) at 20 ° C and Illumination 2500 lux. A five-day culture from one jamb was mixed with 150 ml of Hata agar medium (t = 50 ° C) and poured into Petri dishes. After solidification of the agar with a cork drill, wells with a diameter of 4 mm were made, in which 100 μl of the bacterial suspension (working culture) obtained on the basis of the bacterial strain Bacillus spp. KR-083, as well as on the basis of a strain taken as a prototype of Bacillus subtilis VNIISCHM 128 - producer of the biological product Fitosporin.

As a control, we used a nutrient medium — the cultivation medium of the Bacillus spp strain. KR-083. Inoculated plates were placed in the light, and the diameter of the colonies was counted on day 5 of incubation.

Data on the growth-promoting activity of the strain of Bacillus spp. KR-083 and a strain taken as a prototype of Bacillus subtilis VNIISCHM 128 - producer of the biological product Fitosporin, which was determined by the two methods described above, are presented in table 4.

Table 4.
Growth-stimulating activity of rhizobacteria isolated from rice roots, determined by biotests with chlorella and method of stimulating the roots of corn seedlings Strain Chlorella growth after 5 days (⌀, mm) The growth of the roots of corn seedlings (% of control) * at a dilution of 1: 1 at a 1:50 dilution at a dilution of 1: 100 Bacillus subtilis VNIISCHM 128 (Fitosporin) 0 +7.9 +2.3 +1.2 Bacillus spp. KR-083 (claimed) 3.0 ± 0.2 -20.4 +26.3 +11.1 * - average of ten replicates (measurements)

From the data shown in table 4, it is seen that the claimed bacterial strain Bacillus spp. KR-083 has a stronger growth-stimulating activity compared to the prototype Bacillus subtilis strain VNIISKhM 128, in particular in the method of stimulating the roots of maize seedlings with the most commonly used dilution (concentration) of the bacterial suspension (uterine culture of the strain) of 1:50 and 1 :one hundred.

The effect of the bacterial strain Bacillus spp. KR-083 to increase the yield of various crops (potatoes, spring and winter wheat, barley, rice, sugar beets, sunflowers, cabbage, carrots, beets, lettuce, radishes) was experimentally confirmed.

The effectiveness of the strains of Bacillus spp.KR-083 and Bacillus subtilis VNIISKhM 128 in vegetation experiments with radish from Zhara village (during seed treatment and vegetation after germination) is presented in Table 5. In this case, the vegetation experiments were carried out according to the accepted method (Dospekhov, 1986 ), and by "% NPK" we mean agronomically recommended doses of nitrogen, phosphorus and potassium for the active ingredient when growing radishes.

Table 5.
Efficiency of strains of rhizobacteria in vegetative experiments with radish from Zhara Experience option Radish S.Hara Harvest (g / vessel) % increase to control 50% NPK control 5.87 - 100% NPK control 8.05 36.7 Background 50% NPK Bacillus subtilis VNIISCHM 128 + (Fitosporin) 6.83 16,4 Bacillus spp. KR-083 + (claimed) 7.78 22.5 Bacillus subtilis VNIISCHM 128 +++ (Phytosporin) 7.32 24.7 Bacillus spp. KR-083 +++ (claimed) 9.31 58.6 Legend: + - seed treatment; +++ - seed treatment and two treatments for vegetation 7 and 15 days after germination

From the data shown in table 5, it can be seen that in the vegetation field experiments with radish of the village of Heat inoculation with the claimed bacterial strain Bacillus spp. KR-083 contributes to an increase in radish yield, and to a greater extent than similar treatment with the Bacillus subtilis strain VNIISKHM 128 (prototype) -producer of the biological product Fitosporin.

Comparative data characterizing the effect of strains of Bacillus spp. KR-083 and Bacillus subtilis VNIISKHM 128 for increasing the yield of potatoes of the village of Nevsky in production experiments in the Leningrad Region (SE OPO Kalozhitsy) are shown in table 6.

Table 6.
Potato productivity of the village of Nevsky in production experiments in the Leningrad Region (SE OPOK Kalozhitsy) Experience option Productivity (t / ha) Increase in control (c / ha) (%) The control 366.6 - - Bacillus subtilis VNIISCHM 128 (Fitosporin) 387.4 20.8 5.7 Bacillus spp. KR-083 (claimed) 483.3 116.7 31.8

From the data shown in table 6, it is seen that inoculation of the claimed strain of bacteria Bacillus spp. KR-083 in production experiments with potatoes from Nevsky village contributed to an increase in potato productivity to a greater extent than the strain - prototype Bacillus subtilis VNIISKHM 128 - producer of the biological product Fitosporin.

That is, the examples shown in tables 5 and 6 confirm that the inoculation of seed and seedling treatment with the claimed bacterial strain Bacillus spp. KR-083 helps to increase the yield of vegetables by (15-32)%.

Thus, from the data above in the tables it follows that the claimed bacterial strain Bacillus spp. KR-083 has a greater antagonistic activity against phytopathogenic microorganisms (bacteria and fungi), as well as a higher growth-promoting activity of plants due to the ability of biological nitrogen fixation compared to the prototype strain Bacillus subtilis VNIISM 128, and its use in agriculture contributes to an increase in crop productivity .

It is also important that when using the specified strain in the composition of biological products of the bacterium Bacillus spp. KR-083 form spores, which allows you to save these drugs without loss of bacterial viability for a long period, at least 12 months from the date of receipt.

The concentration (number of viable cells and spores) of the bacterial strain Bacillus spp. KR-083 for its effective use in various biological products should be 10 ⁶ -10 ⁸ cells in 1 ml of culture fluid.

It was established experimentally that the use of strain at a concentration of less than 10 ⁶ cells / ml leads to a decrease in antagonistic effect, while increasing the concentration of above 10 ⁹ cells / ml did not lead to increased efficiency of the protective and growth stimulating actions.

The following is an example of obtaining a liquid form of a microbiological preparation based on the inventive strain of rhizospheric bacteria Bacillus spp. KR-083.

1. Pure culture in flasks.

To obtain this culture for bacteria Bacillus spp. KR-083 prepare a liquid nutrient medium of the following composition, g / l:

peptone - 20.0

glycerin - 10.0

K ₂ HPO ₄ - 1.5

MgSO ₄ - 1.5

The pH of the mixture is adjusted before sterilization to a value of 7.0. The resulting liquid nutrient medium is poured into 750 ml rocking flasks of 100 ml and sterilized at 1 atm for 30 minutes. The flasks were inoculated with a sterile microbiological loop from a test tube with beveled nutrient agar (200 g of potato, 10 g of glucose, 20 g of agar per 1 liter of medium) containing a pure bacterial culture of Bacillus spp. KR-083, placed on a rocking chair (220 rpm) and cultivated for 48 hours at a temperature of 28 ° C.

2. Uterine culture in flasks.

The mother culture is grown on the medium and under the conditions specified in paragraph 1 for a pure culture. Sowing flasks produce 5 ml of pure culture and grow 48 hours. This achieves a titer of about 5 × 10 ⁹ cfu / ml (colony forming units in 1 ml).

Seeds (pure and uterine cultures) are stored in the refrigerator for up to 1 month at a temperature of (4-6) ° C.

3. Work culture

A working culture is obtained in bottles or fermenters on similar media, dilution of the nutrient medium by 2 times is allowed. The norm of inoculation with uterine culture is 2-5% of the volume of the medium. The cultivation duration is 48 hours at a temperature of 28-30 ° C. When cultivating working crops, a temperature increase of up to 33 ° C is allowed.

Thus, a concentrate of a suspension of the preparation is obtained.

4. A liquid form of a biological product based on a strain of the bacterium Bacillus spp. KR-083 KR-083.

Due to the relatively high biological activity of the strain used and the high growth rate, the liquid preparation in the finished form, as well as in production, is practically not infected by extraneous microflora. Nevertheless, constant monitoring of all stages of production is necessary.

For preparing the liquid form preparation must be used with an area of 20-40 m ² tiled floors and paved suitable for daily cleaning and sterilization. The room should be kept in excellent sanitary condition, be sufficiently dry and well ventilated. Wet cleaning is performed daily in the room and treated with antiseptics (0.5% solution of bleach, chloramine, phenol or formalin).

The room air is periodically cleaned of microflora by precipitation with sterile water sprayed with a hydraulic console. Before starting work in the room (2-3 hours), the system of bactericidal lamps (BUV, PRK-2 or PRK-4) is turned on. UV lamps are located in the center of the working room above the work tables and under the tables, bearing in mind that the main “dirt” comes mainly from the floor. Air purity is controlled by microbiological samples taken after five minutes of exposure to three Petri dishes with MPA + 0.5% glucose. Cups with seeding are viewed after exposure to a thermostat at a temperature of 37 ° C for 3-5 days. If a large number (10-15 colonies) of microflora is detected in the air of the box, disinfection is performed.

The work is done in clean, starched, well-ironed dressing gowns and caps, separate shoes, which are located in the vestibule of the working room, where workers put them on and take off after finishing work in the room.

The resulting concentrate suspension of the drug based on strain KR-083 (working culture) is diluted with sterile water in a ratio of 1:10 or 1:20 to obtain a final concentration of at least 100 million colony forming units in 1 ml of the drug (at least 10 ⁸ cfu / ml) .

The liquid preparation is sterilely poured into polyethylene bottles or canisters with a capacity of 0.25; 0.5; 1.0 and 10.0 liters. Rinse plastic bottles or cans with alcohol. Container labels and instructions for use are placed on bottles or cans.

The obtained liquid form of the preparation is kept for 3-5 days at 20-25 ° C, after which it can be used for processing crops. Strict control and proper management of the preparation process of the liquid form determine the high quality of the biological product based on strain KR-083 and its corresponding activity in the cultivation of crops.

Claims (1)

The strain of rhizospheric bacteria Bacillus spp. KR-083, deposited under No. 148 VNIISKHM-D, as a means to protect agricultural plants from phytopathogenic microorganisms and stimulate their growth.